Method of tissue repair

ABSTRACT

A method for joining tissue comprising aligning and abutting edges of the tissue to be joined applying a biodegradable, biological solder or an analogue thereof, across the edges and exposing the solder to an energy source under conditions which provide transfer of energy from the source to the solder to cause the solder to bond to the tissue surface adjacent the edges to provide a weld holding the edges together.

TECHNICAL FIELD

The present invention relates to methods for joining living tissues,including veins, arteries, microvessels, tubes, nerves, organ tissuesand biological surfaces, such as peritoneum, omentum, fascia, shin,artificial tissues, and to pharmaceutical products useful in joiningthese tissues.

BACKGROUND ART

Joining tissues such as veins, arteries, microvessels, tubes, nerves,tissues and biological surfaces such as the peritoneum and skin hasmainly been carried out clinically to date by suturing andmicrosuturing.

Microsuturing requires considerable skill and is a time consumingprocedure. Frequently, tissues which have been joined by microsuturingform considerable scar tissue. Some of the difficulties encountered withmicrosuturing can be better understood by considering the example ofrejoining damaged peripheral nerve tissue.

Peripheral Nerves

The electrical signals that control the body's organs and transmitinformation back and forth to the central nervous system (CNS) travelalong peripheral nerves. The structure of these peripheral nerves isanalogous to telephone cables. In a telephone cable there is a strongprotective outer coating that protects all the inner components. Thecopper wires are often grouped in separate insulating tubes that lead todifferent systems. Each of the inner copper wires is a single line thatcan transmit electricity in either direction and has an insulatingcoating around it so that it does not interfere with the lines next toit.

A peripheral nerve (FIG. 1) has an outer membrane consisting ofconnective tissue such as collagen. This membrane (epineurium) protectsand holds the separate nerve bundles together. The nerve bundles whichlie inside this membrane are called fascicles. These fascicles also havea collagen based surrounding membrane and their task is to grouptogether nerve axons supplying a similar area of the body. Inside thefascicle membrane the axons are surrounded by loose connective tissue.The axons are a long extension from a cell body which is containedwithin the CNS in the spine or the brain. Sensory axons transmit to theCNS and motor axons transmit from the CNS. Nerve metabolism is sustainedby the vascular system from both outside the nerve and along the centreof the nerve.

Peripheral nerves can have very small diameters. For instance, themature median nerve at the wrist is approximately 1 cm in diameter andcontains an average of forty fascicles, each of which can contain up to4500 axons. When a peripheral nerve is cut all axons distal to the woundchange their properties as axon flow is cut off from the cell body. Evenwhen the nerve is reconnected, these axons continue to degeneratedistally. The Schwann cells which normally wrap themselves around theaxons as insulation guide regenerating axons. Joining nerves asaccurately as possible by lining up corresponding fascicles enables theaxons to more efficiently regenerate.

Operating upon nerves has been facilitated by using magnification andspecial microsurgical equipment. Accurate repairs need to be effected atthe fascicular level ensuring that regeneration is along the correctbundle leading to the original area those axons supplied. The currenttechnique of peripheral nerve repair uses microsuturing (FIG. 2). Thistechnique requires a dedicated, trained surgeon as microsuturing of justone of the many fascicles with three or more microsutures (using say a70 micron diameter needle and 30 micron thread) can take very longoperating times.

Microsuturing is at present clinically used where the skills areavailable. Unfortunately, there are relatively few surgeons who have thenecessary manipulative skills for operating at high magnification. Evena reasonable microsuturing technique results in long operating timeswith added damage to the inner axons due to sutures penetrating the thininsulating perineurial sheath. The use of sutures results in somescarring of the repair due to foreign body reaction. There is, alsoevidence which indicates that in the long term scar tissue formation andscar maturation can lead to impairment of the joined nerve.

Work has been performed on the use of lasers alone in effecting nervejoins. One of the problems of laser welding has been the fact that theintact gel-like nerve tissue of the axons is actually under pressurewithin the fascicle. When the fascicle is cut this material extrudes.This can lead to the direct laser weld being formed on nerve tissuerather than the surrounding membrane of the fascicle, causing nervedamage. To date the welds have typically been made using infrared laserssuch as CO₂ lasers which rely on water absorption for energy transfer.Tissue preparation before welding relies on overlapping the nervemembranes. This is difficult due to the extruding gel-like axons and socan lead to denaturation of the nerve axon material. The affected tissuetends to scar and the fibrous tissue that proliferates as a result is apoorer electrical conductor than nerve tissue. The bonds formed to dateas described in the prior art using laser welding have typically lackedstrength. These laser joins alone tend to fail so microsuturing has beenused in addition to welding to strengthen these joins.

To deal with at least some of the deficiencies of laser welding, variousglues have been used in forming the welds. These low proteinconcentration, fluid glues tend to run between the ends of the nervethat are being joined which may result in damage to the axoplasm of thenerve fascicle and also hinder regeneration. They are also appliedaround the join which is then circumferentially welded. These joinslater show thick scarring which causes stricture of the nerve. Moreover,the joins tend to be weak.

The welding techniques so far available also tend to lack precision.Factors that influence the precision of this approach adversely includedifferences in: the consistency of the glue used; the aperture of theneedle or other device used to apply the glue; and the pressure exertedin applying the glue.

DESCRIPTION OF THE INVENTION

The present invention provides a method for joining tissue comprising:

-   -   aligning and abutting edges of the tissue to be joined;    -   applying a solder, across the aligned and abutted edges; and    -   exposing the solder to an energy source under conditions which        provide a transfer of energy from the source to the solder to        cause the solder to bond to the tissue surface adjacent the        edges thus providing a weld holding the edges together.

In addition to causing the solder to bond to the protein of theunderlying tissue, the energy transfer can affect the structure of thesolder itself leading to bonding within the solder and an enhancement ofthe strength of the solder and hence the join.

Drops of solder are typically used where the solder is a fluid solder,and are “painted” across the edges.

The solder can also be provided as a preformed solid strip.

The energy source is typically a laser.

A variety of tissue types can be joined using this method. The method isapplicable to anastomoses of biological tubes including veins, arteries,lymphatics, nerves, vasa efferentia, fallopian tubes, bile ducts, tubesof the alimentary canal, the ureter, the urethra, tear ducts, bronchiand any other such bodily tubes as well as to repairs of incisions ortears of biological organs such as kidneys, liver or spleen, or ofbiological surfaces such as the peritoneum and skin. It will thereforebe understood that the method can be used in a variety of joinsituations including the joining of cylindrical anastomoses and theclosure of linear defects such as incisions.

Where the tissue repair is with respect to nerve tissue or other tissuetubes where the tube contents need to be protected from damage, it isespecially important that the weld should not be concentrated on theedges being joined as this can damage extruded tissue. Rather, the weldshould be distributed across the planar or tubular surface in which thediscontinuity lies.

Where the tissue to be repaired is an essentially hollow body tube suchas a blood vessel, the repair can additionally comprise the insertion ofa thin-walled hollow cylinder of solder inside the tube under repair sothat the cylinder spans the severed portions of the tube. Typically,while the severed tube and cylinder assembly is held together, energyfrom the energy source is directed through the tube wall to bond thecylinder to the tube ends. The cylinder may incorporate a dye, ashereinafter described, to attract energy to the cylinder for moreefficient welding. The repair is completed by the application of atleast one strip or drop of solder across the edges on the outer surfaceand treating the applied solder as described above.

Where the repair is with respect to tissue surfaces such as peritoneum,it will be understood that it is less important to avoid concentrationof welding on the edges.

The method can also be modified for the repair of other discontinuitiesin tissue surfaces such as holes, resulting from accident or surgery. Inthis form of the invention the solder may be spread or pre-cut toconform to the shape of the repair site, and the edges of the repairsite may not need to be aligned or abutted for the repair to beeffected.

A typical nerve repair using the method of the invention is one in whichthe edges are ends of a cut peripheral nerve fascicle that are to bejoined together or an end of a nerve fascicle and the fascicle ofsubstitute nerve graft material. This latter situation is particularlyapplicable where nerve repair is required but a section of the nerveunder repair has been severely damaged or is unavailable, so that theavailable ends of the fascicle are too remote from each other to bedirectly joined. The actual nature of the damage sustained by the nerveand whether the repair is a primary or secondary repair are factorsaffecting recovery but in any case the edges of nerve fascicles to bejoined are cleanly cut at right angles prior to joining.

Application of the solder as a strip or strips, with space between fornatural co-aptation of the surfaces themselves permits the nerve underrepair to revascularise. Circumferential welding, by comparison, caninhibit the body's natural healing process and so slow down bloodcapillary access needed for the area of repair. Laser soldering andsuturing techniques ultimately rely on the body regenerating connectivetissue to hold the nerve together after either solder or sutureconnections break down and are replaced by the healing process. Thepresent inventors have shown in in vivo experiments that successfulregeneration can be achieved by the methods of the present inventionwithout restriction on surrounding tissue movement after the operation.In the case of nerve repair operation on human patients it is routine toinitially restrict the movements of the joints of the operated limbs toassist in reducing tension across the repair site.

Typical biodegradable, biological solders useful in the method of theinvention include protein solders.

It is envisaged that other naturally occurring biomolecules could beused as alternatives. Further analogues of biological, biodegradablepolypeptides could be used. Analogues of biological, biodegradablepolypeptides useful in the invention include synthetic polypeptides andother molecules capable of forming a viscous “glue” that does not reactadversely within the tissue undergoing repair.

The protein solder may be a solid or a fluid solder composition.

Fluid protein solder compositions useful in strip welding typicallycomprise between 100 and 120 mass % of protein relative to water.Preferably, fluid protein solders comprise between 100 and 110 mass %protein relative to water.

The fluid solder strip is typically 50 to 200 μm in thickness. Itslength is selected to suit the join to be formed but typically is of theorder of 2 to 3 mm in length. It is typically painted across the join.

Solid protein solder compositions useful in strip welding typicallycomprise between 120 and 230 mass % protein relative to water.Preferably the strip comprises 170 to 230 mass % protein and morepreferably about 210 mass %.

It will be understood that different proteins will have differentdegrees of solubility in water or appropriate solutions which in turnwill affect the optimum concentration of protein in the composition fordifferent protein solders. Appropriate ranges for particular proteins inboth solid and fluid solders can be determined based on the knownproperties of the proteins.

Typically, the solid protein solder composition is provided as apreformed strip. Solid solder strips are easier to manipulate than fluidsolders. Under the moist conditions inherent in surgery fluid soldersmay run making it difficult to laser denature the solder before it hasspread. The solid solder strips can have a paste like or more rigidconsistency. They are typically placed across the join withmicroforceps. In one form of the invention, it is envisaged that thesolder strips will be substantially rectangular in shape. However,different shape strips may be required in different repair situations.It may also be desirable to provide a plurality of strips joinedtogether for efficient repair of a large or a substantial number ofrepair sites.

The protein solder may comprise a single protein of which albumin is atypical example or alternatively the solder may comprise more than oneprotein.

Albumin has desirable qualities for solid solder strip formation sinceit has a high proportion of β sheet structure which gives rigidity tothe strips. Fibrin is another example of a protein with significant βsheet structure. Incorporation of α helical protein in the solder canassist in making the strips more malleable and thus retain a flatterprofile which is particularly well suited for joining nerve ends. Anexample of a suitable proportion of α helical protein is between 1 and10% by weight of the protein used. About 5% is a preferred amount.Collagen, tropoelastin and elastin are examples of suitable α helicalproteins.

Protein used in the solder is selected to minimise the risk of adversehost reactions and should therefore preferably be an autologous proteinfor the host or a foreign protein of low antigenicity.

The proteins may be obtained from any suitable source. Recombinantly orsynthetically produced proteins as well as purified naturally occurringproteins may be used.

Preferably, when the solder is to be used with a laser which producesenergy at a suitable wavelength the composition includes a substance,such as a dye, which absorbs energy at the wavelength produced by thelaser with which the solder is to be used. It is preferable to choosethe combination such that the dye or other substance absorbs the energytransmitted by the laser efficiently but the underlying tissue to bejoined absorbs the transmitted energy poorly. The dye or other substanceassists in making the welding specific to the solder used which in turnassists in minimising accidental tissue heating damage to the underlyingtissue.

The process of bonding, where protein solders are used, relies onprotein molecules being available for cross-linking. This occurs whenthe protein molecules are unfolded. Upon laser irradiation of, forinstance, an albumin and indocyanine green containing solder at a nervetissue join, albumin molecules are heated through energy transfer fromthe indocyanine green molecules, allowing them to unfold and bondbetween themselves and to neighbouring tissue surface such as thefascicle membrane.

Dyes which contrast with the tissues being repaired can also be usefulin making the solder easier to see. An example of a dye with thisproperty is indocyanine green.

When the laser used is a CO₂ laser, a dye will not assist the energytransfer, as the energy transfer is by water absorption.

The energy provided by the energy source should be sufficient to bondthe solder to form the weld while minimising damage to the underlyingtissue. The temperature required to denature a protein solder istypically at least 50° C. and may exceed 100° C. A preferred range is50° to 90° C. A particularly preferred range is 80° to 90° C.

The time of treatment for each join to be effected can vary depending onsuch factors as ambient conditions, altitude, and of course the natureof the tissue to be joined. The duration of treatment is typicallyshort. A 30 second passage for laser treatment of a 0.4 mg strip is anexample of the time involved although it will be understood that shorteror longer treatment times could be required. It will be understood thatsolid solder takes longer to denature than fluid solder.

In a second aspect the present invention provides a protein soldercomposition comprising protein and a suitable solvent for the protein.Water is typically used as the solvent for water soluble proteins.

In a third aspect the present invention provides a kit for use injoining tissues comprising, in a preferably sterile pack, a plurality ofprotein solder strips and/or shapes of the second aspect of theinvention. Preferably a plurality of strip lengths and/or shape sizesare included in the pack.

The kit preferably includes means for sterile manipulation of thestrips. The kit also preferably includes means for measuring the strips.

The kit may also comprise an energy source such as a fibre coupled lasersystem.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of a peripheral nerve in schematic form.

FIG. 2 shows the joining of a peripheral nerve by prior artmicrosuturing techniques.

FIGS. 3 a) and b) shows in schematic form joining of a nerve fasciclewith a) fluid solder and b) solid strips.

FIG. 4 shows the repair site of a 0.3 mm diameter tibial nerveimmediately after: a) diode laser strip welding, and b) microsuturing.

FIG. 5 a shows a rat tibial nerve welded by the laser solder methods ofthe present invention. The solder and the membrane are denatured but nosignificant change to the axons has occurred (x100, Giemsa).

FIG. 5 b shows a rat sciatic nerve joined by microsuturing using 10-0nylon. Localised perineural and axonal damage occurs.

FIG. 6 shows in schematic form joining of a blood vessel using internalbiodegradable solid solder cylinder and external solid solder strips.

FIG. 7 shows in schematic form a cross-section of a repaired nervefascicle.

FIG. 8 shows the method used for measuring tensile strength of repairednerves.

FIG. 9 shows a solid solder strip positioned upon a severed rat tibialnerve just prior to laser welding.

FIG. 10 a shows regeneration of myelinated axons in a laser nerve repairthat has regenerated for 3 months.

FIG. 10 b shows fibrous tissue around a suture in a sutured nerve thathas regenerated for 3 months.

FIG. 11 shows muscle action potential results for repaired nerves.

BEST METHOD OF CARRYING OUT INVENTION

Tissue repair is performed using a laser to activate a protein solderapplied across the tissue edges to be joined. This solder denatures uponlaser irradiation and bonds with itself and the neighbouring membrane toform the join. The procedure is shown schematically in FIGS. 3 and 7 fora repair to a nerve fascicle. The solder is applied in longitudinalstrips across the join.

Nerve Repair

Repair to severed nerve tissues is effected by the placement of solderacross the severed edges and exposure of the solder to laser asdescribed above. In order to repair nerve tissue without damage to thecontents of the nerve it is desirable to avoid concentrating the weld onthe edges as extruded nerve contents may be damaged. Rather the weldshould be distributed across the planar or tubular surface in which thediscontinuity lies.

Hollow Body Tube Repair

When repairing hollow body tubes it is preferable to insert an internalcylinder of solder into the tube so that it lies between thediscontinuity. The severed ends of the tube are placed over oppositeends of the solder cylinder. The arrangement is shown in FIG. 6.Lasering can then be effected to cause bonding of the cylinder to thetube being joined while the arrangement is held in place. If there is agood fit between the tube and the cylinder this laser step may not berequired. The join is completed by the addition of external solder asfor nerve repairs.

Tissue Surface Repairs

Surfaces such as peritoneum are planes of tissue in which joins withoutsutures can be achieved by the application of solder across thediscontinuities to be joined and welding as described above. In thiscase it is less important to avoid concentration of welding on theedges.

Laser and Solder System Suited to Nerve Fasicular Repair

To denature the protein solder, a GaAs/GaAlAs laser diode with a nominalpower of 250 mW (Spectra Diode Labs, San Jose, Calif.) is used. Thelaser light is coupled into a 100 μm diameter core optical fibre whichis hand held in a fibre chuck. The diode is operated in continuous modeat 75 mW during the laser soldering. Because this laser is Class 3b, andis not eye safe, protective glasses must be worn at all times when usingthis laser.

A suitable protein solder is a mixture of water, albumin and indocyaninegreen (ICG) dye (Becton Dickinson, Mo.). Indocyanine green has a maximumabsorption coefficient at a wavelength of 805 nm of 2×10⁵ M⁻¹ cm⁻¹. Thepercentages of albumin and dye compared to the water were 110% and 0.6%respectively for fluid solder. 210% albumen was used in preparing solderstrips. It is notable that ICG dye appears to preferentially bind withthe albumin ensuring that heat is efficiently transferred to denaturethe protein solder.

Laser Soldering Technique

When conducting the surgery an operating microscope or some form ofmagnification is preferable. For a laser solder repair of a tubular joina section of thin gauze material is placed under the join to assist in arotation technique. The tissue edges are prepared in accordance withstandard techniques for the tissue type and geometry of the repair.

Using micro forceps the edges are aligned and butted together. A 2 mmlong stripe of fluid solder is “painted” longitudinally across thejunction of the edges using a 30 gauge needle freshly coated in thesolder. Alternatively a strip solder is laid across the join usingmicroforceps. The solid strip repair method is simpler. A solid strip isheld in special microforceps and placed across the junction parallel tothe length of the structures to be joined. The laser output is thendirected at the solid strip and the solid solder changes coloursignalling denaturation which causes it to adhere to the underlyingtissue membrane. The process is repeated with further strips to ensure astrong union of surface.

The diode laser output from the 100 μm optical fibre is then used in a30 second continuous pass to denature solid solder into a strip weld. Ata diode output power of 75 mW, the solid solder strip turns brown on thesurface and opaque underneath from the single pass, signallingdenaturation. When using fluid solder denaturation occurs more quickly.A two second laser pass can be sufficient to denature the fluid solder.Generally a second layer of fluid solder is applied to the strip inorder to increase the strength of the weld and the two second laser passis repeated. The gauze under the join is then used with themicro-forceps to rotate the join so that other strips can be applied.

Preparation of Fluid Protein Solder

Composition:

-   -   Albumin (fraction V powder from Sigma, St. Louis, Mo.) at least        100% to 110% by weight compared to water.    -   Indocyanine Green (Becton Dickinson, Mo.) approximately 0.6% by        weight compared to water.    -   Water (injection grade)

Procedure: A solution of ICG in water was prepared in a minitube. Thealbumin was added to the tube. The albumin and solution were mixed usinga vortex mixer. This causes the protein structure to change leading tolinkage of protein molecules to each other rather than to watermolecules.

Preparation of Solid Protein Solder

Composition:

-   -   Albumin (fraction V powder from Sigma, St. Louis, Mo.) 210% by        weight compared to water    -   Indocyanine Green (Becton Dickinson, Mo.) approximately 0.6% by        weight compared to water    -   Water (injection grade)

Procedure: The ICG was dissolved in the water and the albumin was addedto this solution in a minitube. This combination was mixed using avortex mixer and a needle. The combination was mixed (for approximately3 minutes) until it became a homogenous, malleable, green paste. Thephase of the mixture changed under this mixing technique to provide analmost solid composition with mainly protein to protein linkages ratherthan protein to water linkages. The system is no longer a solution atthis stage. The protein paste was malleable and could be cut into stripsfor up to about 30 minutes after mixing. After this time the pastehardened due to dehydration and became too hard to cut.

The resulting strips were between 50 and 100 μm in thickness, about 0.6mm wide and 1.5 to 3.5 mm long. It will be understood that where thestrips are used in mending nerve fascicles that the desired width andlength are dictated by fascicle dimensions. The width, thickness andlength mentioned here are those suitable for use with a rat tibial nervewhich has a diameter of 0.2 to 0.8 mm. The ratio of strip width to nervecircumference is typically:

-   -   Width ˜1/5 circumference

EXAMPLE 1

A 100 μm core optical fibre-coupled 75 mW diode laser operating at awavelength of 800 nm has been used in conjunction with a protein solderto stripe weld severed rat tibial nerves, reducing the long operatingtime required for microsurgical nerve repair. Welding is produced byselective laser denaturation of the protein based solder which containsthe dye indocyanine green. Operating time for laser soldering was 10±5min. (n=24) compared to 23±9 min. (n=13) for microsuturing. The lasersolder technique resulted in patent welds with a tensile strength of15±5 g, while microsutured nerves had a tensile strength of 40±10 g.Histopathology of the laser soldered nerves, conducted immediately aftersurgery, displayed solder adhesion to the outer membrane with minimaldamage to the inner axons of the nerves. An in vivo study, with a totalof fifty-seven adult male wistar rats, compared laser solder repairedtibial nerves to conventional microsuture repair. Twenty-four lasersoldered nerves and thirteen sutured nerves were characterised at threemonths and showed successful regeneration with average Compound MuscleAction Potentials (CMAP) of 2.4+\−0.7 mV and 2.7±0.8 mV respectively.Histopathology of the in vivo study, confirmed the comparableregeneration of axons in laser and suture operated nerves. A faster,less damaging and long lasting laser based anastomotic technique ispresented.

Materials and Methods

1. Animals

A total of fifty-seven young adult male Wistar rats weighing between 400and 550 g at the outset were used in this study. Thirty four ratsreceived laser solder repair and the remaining twenty-three receivedstandard microsuture repair as detailed below. Five rats of each repairmethod were used for tensile strength measurements and light microscopyimmediately after surgery and the remaining thirty-seven rats weresubjected to a study of functional recovery using electrophysiology andhistopathology.

2. Laser Solder System

To denature the protein solder, a GaAs/GaAlAs laser diode with a nominalpower of 250 mW (Spectra Diode Labs, San Jose, Calif.) was used. Thelaser light was coupled into a 100 μm diameter core optical fibre whichwas hand held in a fibre chuck. The diode laser was mounted on a heatsink, and the diode current and temperature were controlled by a SDL-800diode driver. The diode was operated in continuous mode at 75 mW duringthe laser soldering, corresponding to a maximum power density of 955W/cm at the tissue. The laser output power was measured with a Scientech(Boulder, Colo.) power meter. Because this laser is Class 3b, and is noteye safe, protective glasses were worn at all times when using thislaser.

The solder used in this study was an albumin based protein mixture, alsocontaining indocyanine green (ICG) dye (Becton Dickinson, Mo.).Indocyanine green has a maximum absorption coefficient at a wavelengthof 805 nm of 2×10⁵ M⁻¹ cm⁻¹. It is notable that this dye appears topreferentially bind with the proteins ensuring that heat is efficientlytransferred to denature the protein solder.

3. Surgery

Anaesthesia was maintained during surgery using a mixture containingFluothane (4% during induction, 2% thereafter) in O₂ (1 L/min). Using aOPMI 7 operating microscope (Zeiss, West Germany) the sciatic nerve ofthe left leg was exposed at the sciatic notch so that the nerve branchescould be distinguished. The tibial branch, just below the sciatic notch,was exposed from the surrounding subcutaneous tissue for a length of 1cm. For a laser solder repair, a section of thin gauze material wasplaced under the tibial nerve to assist in rotation of the nerve, andfor the suture repair, a section of plastic was placed under the nerveto allow easier suturing. The tibial nerve was then severed withserrated micro-scissors and left for 3 minutes for the normal extrusionof axoplasm to occur. This was then trimmed with the serratedmicro-scissors as required, after which the nerve was repaired witheither four laser solder strips or four 10-0 perineurial sutures.

The laser solder method involved aligning both stumps of the severednerve with micro-forceps then a 2 mm long strip of solder was “painted”longitudinally across the junction of the severed ends using a 30 gaugeneedle freshly coated in the solder (FIG. 3 a). The diode laser outputfrom the 100 μm optical fibre was then used in a continuous two secondpass to denature the solder into a strip weld. At a diode output powerof 75 mW, the solder was observed to turn brown on the surface andopaque underneath from the single pass, signalling denaturation. Asecond layer of solder was applied to the strip and the two second laserpass was repeated. The gauze under the nerve was then used with themicro-forceps to rotate the nerve so that three other two layeredstripes could be applied, each approximately 90° apart.

Seven rats were operated with a more advanced version of the organicsolder, which is still an albumin based protein mixture but it has theadvantage to be dehydratated and cut into solid rectangular strips (FIG.9). The average surface area of the solder strips was 1.5+\−0.5 mm² andthe thickness was 0.15+\−0.01 mm. Four strips were positioned along thetibial anastomized nerve and then radiated with the same procedureadopted for the fluid solder. The solid strip was fused with theperineurium of the tibial nerve by the laser radiation, joining theextremites of the sectioned nerve.

For all operation the time of anastomosis was recorded and aphotographic record was taken for later reference. The animals wereplaced in their cages with no restriction of movement for 3 months.

4. Immediate Measurement of Tensile Strength and Histopathology

In ten of the operated rats, the 1 cm long section of the laser andsuture repaired nerves was harvested immediately for tensile strengthmeasurements. Fine silk was tied to each end of the tibial nerve. Oneend was then attached to a calibrated force transducer (FT30C, GrassInstruments, Quincy, Mass.) and the other to a screw driven translator(FIG. 8) As the screw was turned the translator would stretch the nervein a slow and steady manner. The applied tension was observed on anoscilloscope connected to the output of the force transducer. Tensionwas applied until the nerve separated, and the breaking force wasrecorded. The nerves were kept moist, as upon drying, the tensilestrength can be increased.

For light microscopy the anastomosis site of the tibial nerves werefixed in 5% formalin, alcohol dehydrated, imbedded in paraffin,longitudinally sectioned and stained with either Masson's trichrome orGiemsa.

5. Functional Assessment: Histopathology and Electrophysiology

Three months post operatively the rats were reanaesthetised using themethod described in section 3. The site was exposed and the anastomosisof the tibial nerve observed. The two other branches of the sciaticnerve, the peroneal and sural nerves were then severed so that only thetibial nerve branch of the sciatic nerve could conduct electricalstimulation of the sciatic nerve to the muscles of the hind foot. Twodays later the rats were positioned on their side and insulated from thetable by a folded surgical drape. An. infrared lamp was used to maintaintheir rectal temperature above 36° C.

A clinical electromyograph (Cadwell Sierra EMG/EP) was used forstimulation and recording. Two 25 gauge stimulating electrodes were,placed 10 mm apart on each side of the sciatic nerve above the sciaticnotch, near the hip. The nerve was activated using rectangular pulses(0.1 to 0.3 ms; 0 to 30 mA; 1 Hz). Compound muscle action potentials(CMAPs) were recorded from the plantar muscles of the foot in responseto supramaximal stimulation of the sciatic nerve. A set of threerecording electrodes were used. A 25 gauge ground electrode was insertedsubcutaneously between the stimulating and recording electrodes^(1,2). A30 gauge reference electrode was inserted into the heel pad and a 30gauge recording electrode was inserted into the plantar muscles of thefoot. The CMAPs were recorded and processed to determine their negativewave peak value.

Histopathology of the sutured and laser soldered nerves, was conductedafter the Electrophysiology test with the same procedure as adopted insection 4.

Results

At the completion of surgery all anastomoses were successful. Theoperating procedure was found to be easier for laser soldering than formicrosuturing. This resulted in the shorter operating times for lasersolder repairs {10±5 min (n=24)} than {23±9 min (n=13)} for microsuturerepairs. The tensile strength of five laser solder repaired nervesimmediately after the operation was 15±5 g and the tensile strength ofthe microsutured nerves, 40±10 g.

Histopathological examination of the anastomosis sites immediately aftersurgery demonstrated that the albumin and ICG dye based laser solderdoes bond well with the outer membrane of the nerve, the perineurium,while the inner axons remain unheated. In FIG. 10 a, a tibial nervefascicle weld produced by the diode laser and albumin/ICG dye solder isshown in section. Both the protein solder and the perineurium havedenatured forming the bond. On the lower side of the bond, the axoplasmhas its normal wavy structure. Note that since heating is concentratedat the dye, only denaturation of the solder and adjacent perineuriumoccurs.

One of the promising aspects of laser anastomosis is the potential forreduced damage to the axoplasm by removing the need for sutures. Asection showing the effect of microsuturing nerve fascicles using10/0-nylon is shown in FIG. 5 b. This section stained with Giemsa,displays axon extrusion at the join, as well as localised perineurialand axonal damage due to the suture.

Histopathology at 3 months shows regeneration of myelinated axons inlaser nerve repairs (FIG. 10 a), with no discontinuity of either thefibers and their sheaths, or the fibrous perineurium. No evidence isseen of inflammation or myelin phagocytosis. Full restoration, asassessed by light microscopy, of the histologic integrity of the tibialnerve has been achieved by the laser weld.

The sutured nerves also show successfull anastomosis with myelinatedaxon regeneration, however, it is still evident that the nylon thread issurrounded by fibrous tissue, which creates an obstacle to thedirectionality of the regenerated axons (FIG. 10 b).

The electrophysiological measurements of the in vivo study wereperformed on twenty-four laser solder repaired rats and thirteenmicrosuture repaired rats having three months recovery. Of this groupall twenty-four laser solder anastomoses were patent as were thethirteen microsuture anastomoses. The average amplitude of the muscleaction potentials, resulting from supramaximal stimulation of the nerveabove the repair site was 2.4±0.7 mV for the twenty-four laser solderedtibial nerves and 2.7±0.8 mV for the thirteen microsutured nerves. Thenormal muscle action potential produced by stimulating the tibial nervesupramaximally was recorded at 8.7±3 mV from ten rats (FIG. 11).

Discussion

Clinically, when a major peripheral nerve is severed, forty or morefascicles may need to be individually rejoined. With three or fourmicrosutures per fascicle, suturing tends to be prolonged, as it must bemeticulous. In a nerve graft, where two anastomoses are needed, thesuturing time is doubled. We have sought a suitable method of nerveanastomosis that could at least duplicate the end result but wassignificantly faster than the present hand sewing microsuture technique.A bonus of the described laser soldering method was the demonstratedlack of change to the axonal components beneath the denaturedperineurial layer seen immediately after surgery. Three months latercomparable regeneration was demonstrated by electrophysiological nerveconduction studies.

Industrial Applicability

The present invention has application in the field of surgery where itis of application in joining together tissue edges, in end to end, sideto end and side to side applications.

References

-   1) R. Malik, S. Ho and D. B. Church: A new method for recording and    analysing evoked motor potential from dogs. Journal of Small Animal    Practice (1989) 30, 13-19.-   2) R. Malik, S. Ho: Motor nerve conduction parameters in the cat.    Journal of Small Animal Practice (1989) 30, 396-400.-   3) Laser activated protein bands for peripheral nerve repair. A    Lauto, R Trickett, R Malik, J Dawes, E Owen. European Biomedical    Optics Week—BIOS Europe 195 12-16 Sep. 1995 (Proceeding in Press)

1. A method for joining tissue comprising: aligning and abutting edgesof the tissue to be joined; applying a biodegradable, biological solderor an analogue thereof, as herein defined, across the edges as one ormore transverse strips; and exposing the solder to an energy sourceunder conditions which provide transfer of energy from the source to thesolder to cause the solder to bond to the tissue surface adjacent theedges to provide a weld holding the edges together, wherein when morethan one strip is applied, the strips are spaced apart to permit naturalco-aptation of the join.
 2. The method of claim 1 wherein the tissue isnerve tissue and the edges are ends of a peripheral nerve fascicle or anerve fascicle and nerve graft material, and welding is not effectedalong the line of discontinuity so as to protect the nerve tissue fromdamage.
 3. The method of claim 1 wherein the tissue is an anastomosis ofa biological tube including veins, arteries, lymphatic, vasa efferentia,fallopian tubes, bile ducts, tubes of the alimentary canal, the ureter,the urethra, tear ducts or bronchi, and wherein a hollow cylinder of thesolder is inserted into the tube between the discontinuous ends prior tothe application of solder to the external surfaces of the tube beingjoined.
 4. A method according to claim 3 wherein the discontinuous endsare held in place while energy is applied to the cylinder within thetube to cause the cylinder to bond with the inner surface of the tube.5. The method of claim 1 wherein the tissue is a repair of an incisionor tear of a biological organ including kidneys, liver or spleen, or ofa biological surface such as the peritoneum or skin.
 6. A method forrepairing a discontinuity in a tissue surface comprising: applying abiodegradable, biological solder or an analogue thereof as hereindefined to the discontinuity; and exposing the solder to an energysource under conditions which provide transfer of energy from the sourceto the solder to cause the solder to bond within itself and to thetissue surface to provide a weld holding the solder and tissuessurrounding the discontinuity together.
 7. The method of claim 1 or 2wherein a first strip of the solder is applied and exposed to the energysource, then a second strip is applied close to the first strip andexposed to the energy source and this process is repeated to provide aplurality of strip welds.
 8. The method of any one of claims 1 to 7wherein the biodegradable, biological solder is a protein solder.
 9. Themethod of claim 8 wherein the protein solder is a solid or a fluidsolder.
 10. The method according to any one of claims 1 to 9 wherein theenergy source is a laser.
 11. A method according to any one of claims 1to 10 wherein the solder incorporates a substance which absorbs theenergy from the energy source highly compared to the tissue.
 12. Amethod according to claim 11 wherein the substance is a dye.
 13. A fluidprotein solder composition comprising 100 to 120 mass % protein relativeto water as a starting amount prior to mixing, and a suitable solventfor the protein.
 14. A fluid protein solder composition according toclaim 13 comprising 100 to 110 mass % protein relative to water as astarting amount prior to mixing, and a suitable solvent for the protein.15. A substantially solid protein solder comprising 120 to 230 mass %protein relative to water as a starting amount prior to mixing, and asuitable solvent for the protein.
 16. A substantially solid proteinsolder comprising 170 to 230 mass % protein relative to water as astarting amount prior to mixing, and a suitable solvent for the protein.17. A substantially solid protein solder comprising 210 mass % proteinrelative to water as a starting amount prior to mixing, and a suitablesolvent for the protein.
 18. A kit for joining tissues comprising, in apreferably sterile pack, a plurality of strips and/or shapes of aprotein solder according to any one of claims 15 to
 17. 19. A fluidprotein solder composition comprising 100 to 120 mass % protein relativeto water as a starting amount prior to mixing, and a suitable solventfor the protein, when used in a method according to any one of claims 1to
 10. 20. A fluid protein solder composition according to claim 19further comprising a substance which absorbs the energy from the energysource highly compared to the tissue.
 21. A fluid protein soldercomposition according to claim 20 wherein the substance is a dye.
 22. Asubstantially solid protein solder comprising 120 to 210 mass % proteinrelative to water as a starting amount prior to mixing, and a suitablesolvent for the protein, when used in a method according to any one ofclaims 1 to
 10. 23. A substantially solid protein solder according toclaim 22 further comprising a substance which absorbs the energy fromthe energy source highly compared to the tissue.
 24. A substantiallysolid protein solder according to claim 23 wherein the substance is adye.
 25. A substantially solid protein solder according to claim 15wherein the protein is albumin.
 26. A fluid protein solder compositionaccording to claim 13, wherein the protein is albumin.
 27. Asubstantially solid protein solder according to claim 15 wherein theprotein is one having a high proportion of β sheet structure.
 28. Asubstantially solid protein solder according to claim 27 wherein theprotein has less than about 10% by weight α helical content.
 29. Asubstantially solid protein solder according to claim 15 with theproviso that the composition is not one consisting of 70.3% by weightcollagen, 16.9% by weight plasticizer and 9% by weight water.